3 replies to this topic

[KenGoh]

Hi guys,

I have tried to run a standardcurve on qPCR but it seems like not all contain my samples which are cDNA i reverse transcriped. besides, it seems it's a late amplification. I'm sure i've loaded the samples and with that concentration the cT value should be high. couldn't figure out the problem. Mind cek out the results i attached along.

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[doxorubicin]

Cts are high, not low.  This means your samples are too dilute.  Take the finished reaction (Wells A1 and A2 and wells E1 and E2), purify the product using Qiaquick PCR purification.  Dilute this 1:10, 1:100, 1:1000, 1:10000, 1:100000 in TE buffer and some of these should be in the good range for a STD curve.

[KenGoh]

Opps. mistake it's too high. sorry. Thanks a lot got the help. will give a try. This as the extracted RNA from samples. i only have the most 200ng/ul concentration is a 50uL reaction volume. If my target samples were that low the quantification will result in low cTs also rite ?

Edited by KenGoh, 08 January 2013 - 07:33 AM.

[doxorubicin]

Most RT kits recommend 1ng to 5ug of total RNA, so you have plenty of RNA.  Just follow the RT protocol carefully and you will probably have to dilute the finished reaction at least 1:10 to be in a good range for qPCR.  To generate the standard curve, this thread may be helpful:  http://www.protocol-...standard-curve/